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Abstract. – OBJECTIVE: MicroRNA-329-3p (miR-329-3p) has been shown to be involved in tumor development. But its role in hepatocellu-lar carcinoma has not been explored. Our study aims to explore the effect and mechanism of miR-329-3p on hepatocellular carcinoma devel-opment.

PATIENTS AND METHODS: Hepatocellu-lar carcinoma tissues and paired paracancer-ous specimens from 31 hepatocellular carcino-ma patients undergoing surgery were collect-ed. Quantitative real-time polymerase chain re-action and Western blot were employed to mea-sure genes expression at mRNA and protein lev-el. CCK-8 and transwell assays were performed to evaluate hepatocellular carcinoma cells pro-liferation and migration. Dual-Luciferase report-er gene assay was designed to validate the tar-get gene of miR-329-3p.

RESULTS: Our study showed miR-329-3p ex-pression was significantly lower in hepatocel-lular carcinoma tissue. MiR-329-3p mimic inhib-its proliferation and migration of HepG2 cells. By using Dual-Luciferase reporter gene assay, we proved that miR-329-3p inhibited HepG2 cell proliferation and migration by targeting USP22 directly. By up- and downregulation of USP22 expression, we also proved that USP22 can activate the Wnt/β-Catenin pathway, which in turn affected the proliferation and migration of HepG2 cells.

CONCLUSIONS: We demonstrated that miR-329-3p can inhibit HepG2 cell proliferation and migration by inhibiting USP22-Wnt/β-Catenin pathway. Our study provides novel insights into the aetiology and potential treatment of hepato-cellular carcinoma.

Key Words:MicroRNA-329-3p, USP22, Hepatocellular carcino-

ma, Proliferation, Migration, Wnt/β-Catenin.

Introduction

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Globally, liver cancer is the fourth leading cause of can-cer-related deaths1. Every year, a large number of patients with HCC die, which places a heavy bur-den on medical systems. The 5-year survival rate of liver cancer is only 18% and it is the second fatal tumor after the pancreatic cancer2. The poor prognosis of liver cancer is due to high recurrence and metastasis rates3. The cause of HCC is not fully understood. But its common causes include hepatitis virus infection, alcoholic or non-alco-holic cirrhosis, etc.4. Nowadays, increasing re-searchers are studying the pathogenesis of HCC in order to find a way to improve the treatment effect of HCC.

The role of microRNA (miRNA) in malignant tumors has attracted widespread attention from researchers all over the world5,6. MiRNA is a type of small RNA with a length of 20 to 26 nt. It often targets to the 3 ‘untranslated region (3’UTR) of the target mRNA, which makes the downstream target gene degraded and inactivated7,8.

The discovery of miRNA has completely changed our understanding of cancer’s complex gene networks. MiRNAs play an important role in various biological and pathological process-es including proliferation and metastasis9 and down-regulation of tumor suppressive miRNAs or overexpression of oncogene miRNAs plays a key role in tumorigenesis10-12.

MiR-329-3p was found to play a role as a tumor suppressor13-15. However, there are few reports about miR-329-3p in HCC. This study

European Review for Medical and Pharmacological Sciences 2020; 24: 9932-9939

R.-Q. XIN1, W.-B. LI1, Z.-W. HU1, Z.-X. WU1, W. SUN2

1Department of Hepatobiliary Surgery, Inner Mongolia People’s Hospital, Hohhot, Inner Mongolia, P.R. China2The Postdoctoral Research Station, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P.R. China

Ruiqiang Xin and Wubing Li contribute equally and should be regarded as co-first authors

Corresponding Author: Wei Sun, MD; e-mail: sunwei85@pumch.cn

MiR-329-3p inhibits hepatocellular carcinoma cell proliferation and migration through USP22-Wnt/β-Catenin pathway

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focused on the role of miR-329-3p in HCC and its mechanism.

Patients and Methods

Tissue SampleFrom 2007 to 2019, HCC tissues and paired

paracancerous specimens from 31 HCC pa-tients undergoing surgery were collected. The patients signed an informed consent before the operation. The medical Ethics Committee of the Inner Mongolia People’s Hospital approved the study.

Cell CultureHuman HCC cell lines HepG2 were all cul-

tured in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Carlsbad, CA, USA) with 10% (V / V) FBS (Hyclone, Chicago, IL, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin (GIB-CO, Carlsbad, CA, USA), cultured in an incuba-tor at 37°C, 5% CO2. PNU-74654 (Wnt/β-actin pathway inhibitor, Selleck Chemicals, Shanghai, China) is dissolved in dimethylsulfoxide (DMSO; Solarbio, Beijing, China) to make a solution with a concentration of 10 μM.

TransfectionsMiR-329-3p mimic, miR-329-3p inhibitor,

USP22 siRNA, USP22 overexpression plasmids (pcDNA3.1- USP22) were designed and synthe-sized by GenePharma (GenePharma, Shanghai, China). HepG2 cells were seeded on 12-well plate with a density of 5×105 cells/well. Lipofectamine 2000 (Invitrogen, Shanghai, China) was used to transfect miR-329-3p mimic, miR-329-3p in-hibitor, USP22 siRNA, pcDNA3.1- USP22 into HepG2 according to the protocol. Proteins and genes were isolated from HepG2 cells for subse-quent experiments.

Cell Proliferation and Cell Viability Assay HepG2 cells were seeded in 96-well plates at

density of 2 × 103 cells / well, each well was load-ed with 200 μL culture medium. 20 μL of CCK-8 solution (Dojindo Laboratories, Kumamoto, Ja-pan) was added to each well, and after 24 h, 48 h, 72 h, 96 h incubation, the optical density value (OD value) of each well was measured by a mi-croplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm, and the cell proliferation was calculated by the OD value.

Transwell Assay2×104 HepG2 cells were seeded on the surface

of phospholipid carbonate of 12-well transwell plates (Corning, Corning, NY, USA), the culture condition was 37°C, the cells on the membrane were fixed with 1% paraformaldehyde (Solarbio, Beijing, China) after 24 hours, and stained with 0.2% crystal violet solution (Solarbio, Beijing, China). Ten fields were randomly selected under the microscope, and the number of perforating cells was calculated. The experiment was repeat-ed 3 times.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract and isolate RNA. The Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the RNA concentration. PrimeScript RT reagent kit (TaKaRa, Otsu, Shiga, Japan) and TB Green® Premix Ex Taq™ II kit (TaKa-Ra, Otsu, Shiga, Japan) were used for detec-tion of genes according to the manufacturer’s protocol on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). U6 and GAPDH were used as the internal reference. All reactions were in tripli-cate. Relative expression levels were quantified with 2−ΔΔCT method.

The primer sequence is as follows: GAPDH, forward 5ʹ-AATGGGCAGCCGTTAGG AAA-3ʹ and reverse 5ʹ-TGAAGGGGTCATTGATGG-CA-3 ;́ USP22, forward 5ʹ-GG CGGAAGATCAC-CACGTAT-3ʹ and reverse 5ʹ-TTGTTGAGACT-GTCCGTGGG-3 ;́ U6: forward 5ʹ-GCTTCG-GCAGCACATATACTAAAAT-3′ and reverse 5′-CGCTTC ACGAATTTGCGTGTCAT-3′; miR-329-3p: forward 5′-GTGGAACAGACCTGGT AAAC-3′ and reverse 5′-CAAGTGCGAGTCGT-GCAGT-3′ .

Western BlotAccording to the manufacturer’s protocols, the

cells and tissues were lysed with radio immu-noprecipitation assay (RIPA) lysis buffer (Be-yotime, Shanghai, China), and the total protein was extracted. Proteins were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE). The loading amount was 30 μg of protein per well. Then, the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). Then, the PVDF membrane was incubated

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with primary USP22 (Thermo Fisher Scientific, Waltham, MA, USA; dilution rates of 1:5000), β-catenin (Abcam, Cambridge, UK; dilution rates of 1:5000), c-Myc (Abcam, Cambridge, United Kingdom; dilution rates of 1:5000), Wnt3a (Ab-cam, Cambridge, UK; dilution rates of 1:5000) and β-actin (Abcam, Cambridge, UK; dilution rates of 1:5000) antibodies with 5% fat-free milk at 4°C overnight. Then, the membrane was washed in PBS for three times and incubated with anti-mouse horseradish peroxidase binding secondary antibody (Abcam, Cambridge, UK; dilution rates of 1:10000) for one hour. Super ECL Detection Reagent (Thermo Fisher Scien-tific, Shanghai, China) was used to detect protein bands.

Dual-Luciferase Reporter Gene Assay Wild and mutant plasmids of USP22 (pmir-

GLO- USP22-wt, pmirGLO- USP22-mut) con-taining a wild or mutant miR-329-3p binding sites were designed and synthesized by Ge-nepharma (GenePharma, Shanghai, China). HepG2 cells were seeded at a confluence of 70% into a 96-well plate. Transfection of Luciferase reporter gene plasmid and RNA after 24h using Lipofectamine 2000. After co-transfection of the plasmid for 48 hours, discard the medium and wash it once. Then, the Dual-Luciferase Re-porter Assay System (Promega, Madison, WI,

USA) was used to evaluate fluorescence inten-sity changes according to the manufacturer’s protocol.

Statistical AnalysisStudent’s t-test or analysis of variance (ANO-

VA) was used to analyze measurement data. All data are expressed as mean ± SD. p

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than that in normal paracancerous tissues. We further conducted a correlation analysis and found that the two are negatively correlated, the R2 is 0.7541 (Figure 1C).

MiR-329-3p Inhibits Proliferation and Migration of HCC Cells

We further examined the role of miR-329-3p in the HCC cell line HepG2. We transfected miR-329-3p mimic and miR-329-3p inhibitor into HepG2 cells and found that miR-329-3p mimic can increase miR-329-3p expression and miR-329-3p inhibitor can inhibit miR-329-3p expression (Fig-ure 2A). Through further functional experiments, we found that miR-329-3p mimic can inhibit the proliferation and migration of HepG2 cells, miR-329-3p inhibitor promote the proliferation and mi-gration of HepG2 cells (Figure 2B, 2C).

MiR-329-3p Inhibited USP22 Expression by Targeting it Directly

We have predicted that miR-329-3p may di-rectly inhibit USP22 expression through StarBase 3.0. It suggested that miR-329-3p may directly

inhibit the expression of USP22. To prove this, we first transfected miR-329-3p mimic and miR-329-3p inhibitor into HepG2 cells, and then we examined the expression of USP22. We found that miR-329-3p mimic inhibited USP22 expres-sion (Figure 3A). To further demonstrate that miR-329-3p can directly inhibit the expression of USP22, we performed a Dual-Luciferase reporter gene assay. It turns out that pmirGLO- USP22-wt co-transfection significantly reduced the Lucifer-ase activity (Figure 3B).

USP22 Promoted HCC Cell Proliferation and Migration

We have previously demonstrated that miR-329-3p directly inhibits USP22 expression. We need to prove whether miR-329-3p inhibits the proliferation and migration of HepG2 cells through USP22. We further study the function of USP22. We detected the proliferation and migra-tion of HepG2 cells by up- or down-regulating of USP22 (Figure 4A, B). As a result, we found that pcDNA3.1- USP22 can promote the proliferation and migration of HepG2 cells. (Figure 4C, D).

Figure 2. Effects of miR-329-3p on cell proliferation and migration in HepG2 cells. a, MiR-329-3p expression in HepG2 cells treated with miR-329-3p mimic and miR-329-3p inhibitor. b, Proliferation of HepG2 cells treated with miR-329-3p mimic and miR-329 inhibitor. c, Migration of HepG2 treated with miR-329-3p mimic and miR-329-3p inhibitor, scale bar, 50 μm. **: p

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Figure 3. USP22 is a direct target of miR-329-3p in HepG2 cells. a, USP22 expression in HepG2 cells treated with miR-329-3p mimic and miR-329-3p inhibitor. b, Fluorescence of co-transfection of miR-329-3p mimic and pmirGLO-USP22-mut or pmirGLO-USP22-wt. **: p

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USP22 Promoted HepG2 Proliferation and Migration Through Wnt/β-Catenin Pathway

USP22 siRNA inhibited Wnt/β-catenin pathway related protein, β-catenin, c-Myc, and Wnt3a ex-pression and pcDNA3.1-USP22 promoted β-catenin, c-Myc, and Wnt3a expression (Figure 5A). Accord-ing to functional experiments, pcDNA3.1-USP22 promoted HepG2 cells proliferation and this ef-fect was inhibited by PNU-74654 (Wnt/β-catenin pathway inhibitor) (Figure 5B). pcDNA3.1-USP22 promoted HepG2 cells migration and this effect was also inhibited by PNU-74654 (Figure 5C).

Discussion

MiRNAs play an important role in the develop-ment of tumors8. MiR-329-3p is a newly discovered miRNA. At present, there are relatively few re-searches on his function, mostly focusing on tumor research. Chang et al16 found that the low expression of miR-329-3p is related to the poor prognosis of

cervical cancer. Li et al17 on cervical cancer found that miR-329-3p could inhibit cervical cancer by directly inhibiting MAPK1 expression.

MiRNAs can target genes and regulate down-stream signaling pathways, thereby affecting tumor biological behavior. The regulation of miRNAs on target genes is generally achieved through the complementary base pairing of the 5′ end of the target gene. If the target gene is a tumor suppressor or oncogenic factor, then miR-NA is likely to affect tumor progress18. To explore how miR-329-3p inhibits HepG2 cell proliferation and migration, we predict its downstream mRNA through StarBase 3.0. We found that USP22 mR-NA is one of the possible targets of miR-329-3p.

USP22 is a histone-modifying enzyme and is known to function as a histone deubiquitinating component19. USP22 is found to be highly ex-pressed in a variety of tumors. This suggests that USP22 may work as an oncogene. McCann et al20 found that USP22 acts as an oncogene in prostate cancer to regulate cell proliferation and DNA repair. Liu et al21 related to gastric cancer proved

Figure 5. USP22 promoted Wnt/β-catenin signaling pathway. a, USP22 siRNA inhibited β-catenin, c-Myc, and Wnt3a expression and pcDNA3.1-USP22 promoted β-catenin, c-Myc, and Wnt3a expression. b, pcDNA3.1-USP22 promoted HepG2 cells proliferation and this effect was inhibited by PNU-74654 (Wnt/β-actin pathway inhibitor). c, pcDNA3.1-USP22 promoted HepG2 cells migration and this effect was also inhibited by PNU-74654, scale bar, 100 μm. **: p

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that USP22 can promote gastric cancer growth and metastasis by activating c-Myc/NAMPT/SIRT1-de-pendent FOXO1 and YAP signaling. In addition, other studies have found the role of USP22 in HCC. Ling et al22 demonstrated that when TP53 is inactivated, USP22 promotes hypoxia-induced HCC stemness through a HIF1α/USP22 positive feedback loop. Of course, in some tumors, USP22 is also found to be highly expressed and can exist as a tumor suppressor gene. Kosinsky et al23 found that USP22 acts as a tumor suppressor gene in colon cancer by inhibiting mTOR activity.

According to qRT-PCR and Luciferase exper-iments, we found that miR-329-3p can inhibit USP22 expression by directly targeting USP22. From previous studies20,24, we also know that USP22 can affect the proliferation, metastasis, DNA repair, and stemness of tumor cells. We also found that USP22 can promote the proliferation and migration of HepG2 cells.

USP22 can regulate tumor progression by af-fecting Wnt/β-Catenin Pathway in colon can-cer25,26. And Wnt/β-Catenin Pathway had been shown to affect the proliferation and migration of tumor cells27,28. In this research, we verified whether USP22 also affects Wnt/β-Catenin path-way in HepG2 cells. As a result, we found that USP22 can activate Wnt/β-Catenin Pathway, and the activation of Wnt/β-Catenin pathway promot-ed the proliferation and migration of HepG2 cells. This showed that miR-329-3p can affect the pro-liferation and migration of HepG2 cells through USP22-Wnt/β-catenin pathway.

Conclusions

In summary, we demonstrated that miR-329-3p can inhibit HCC cell proliferation and migration by inhibiting USP22-Wnt/β-Catenin pathway. Our study provides novel insights into the aetiol-ogy and potential treatment of HCC.

Conflict of InterestThe Authors declare that they have no conflict of interests.

AcknowledgementsNot applicable.

FundingNo funding was received.

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